The present invention relates to improvements in magnetic separators and methods of separation of magnetic particles from non-magnetic media, having particular utility in various laboratory and clinical procedures involving biospecific affinity reactions. Such reactions are commonly employed in testing biological samples, such as blood or urine, for the determination of a wide range of target substances, especially biological entities such as cells, proteins, nucleic acid sequences, and the like.
As used herein, the term "target substance" refers to any member of a specific binding pair, i.e., a pair of substances or a substance and a structure exhibiting a mutual affinity of interaction and includes such things as cell components, biospecific ligands and receptors. "Ligand" is used herein to refer to substances, such as antigens, haptens and various cell-associated structures, having at least one characteristic determinant or epitope, which are capable of being biospecifically recognized by and bound to a receptor. "Receptor" is used herein to refer to any substance or group of substances having a biospecific binding affinity for a given ligand, to the substantial exclusion of other substances. Among the receptors determinable via biospecific affinity reactions are antibodies (both polyclonal and monoclonal), antibody fragments, enzymes, nucleic acids, Clq and the like. The determination of any member of a biospecific binding pair is dependent upon its selective interaction with the other member of the pair.
Various methods are available for determining the above-mentioned target substances based upon complex formation between the substance of interest and its specific binding partner. Means are provided in each instance whereby the occurrence or degree of target substance/binding partner complex formation is determinable.
In the case of a competitive immunoassay to determine antigen, for example, the antigen of interest in a test sample competes with a known quantity of labelled antigen for a limited quantity of specific antibody binding sites. Thus, after an appropriate reaction period the amount of labelled antigen bound to specific antibody is inversely proportional to the quantity of antigen in the test sample. Competitive assays for antibodies, employing labeled antibodies (typically monoclonal antibodies) rather than labeled antigen, function in an analogous manner. The resulting immune complexes are separated, for example, by immunoabsorption, physico-chemical adsorption or precipitation of either the complexes or unbound antigen. Antibody-bound labeled antigen is then quantified and a standard curve is constructed from known antigen concentrations, from which unknown concentrations of antigen may be determined.
In contrast, immunometric assays for the determination of antigen, commonly known as "sandwich" assays, involve the use of labeled antibodies instead of labelled analyte. In performing an immunometric assay, a sandwich is formed in which the "layers" are: antibody/multivalent (minimally bivalent) antigen/antibody. The amount of labeled antibody which is bound for each complete sandwich complex (antibody/antigen/antibody) is directly proportional to the amount of target antigenic substance present in the test sample. Sandwich assays can be performed in multi-step fashion with polyclonal antibodies or in fewer steps when monoclonals directed to independent antigenic determinants are employed.
In both the conventional competitive immunoassay and the immunometric assay just described, quantitation of the target substance requires a physical separation of bound from free labeled ligand or labeled receptor.
Bound/free separations may be accomplished gravitationally, e.g., by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the target substance. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, U.S. Pat. No. 4,554,088 and Immunoassays for Clinical Chemistry pp. 147-162, Hunter et al. eds., Churchill Livingston, Edinborough (1983). Generally, any material which facilitates magnetic or gravitational separation may be employed for this purpose.
Small magnetic particles have proved to be quite useful in analyses involving biospecific affinity reactions, as they are conveniently coated with biofunctional polymers, e.g., proteins, provide very high surface areas and give reasonable reaction kinetics. Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunologic reagents, having reasonably good suspension characteristics when mildly agitated. Insofar as is known, however, absent some degree of agitation, all of the magnetic particles presently in commercial use settle in time and must be resuspended prior to use. This adds another step to any process employing such reagents.
Small magnetic particles, such as those mentioned above, generally fall into two broad categories. The first category includes particles that are permanently magnetized; and the second comprises particles that become magnetic only when subjected to a magnetic field. The latter are referred to herein as magnetically responsive particles. Materials displaying magnetically responsive behavior are sometimes described as superparamagnetic. However, certain ferromagnetic materials, e.g., magnetic iron oxide, may be characterized as magnetically responsive when the crystal is about 300A or less in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter. See P. Robinson et al., Biotech Bioeng. XV:603-06 (1973).
Magnetically responsive colloidal magnetite is known. See U.S. Pat. No. 4,795,698 to Owen et al., which relates to polymer-coated, sub-micron size magnetite particles that behave as true colloids.
The magnetic separation apparatus/method used for bound-free separations of target substance-bearing magnetic particles from test media will depend on the nature and particle size of the magnetic particle. Micron size ferromagnetic, i.e, permanently magnetized, particles are readily removed from solution by means of commercially available magnetic separation devices, employing relatively inexpensive permanent magnets. Examples of such magnetic separators are the MAIA Magnetic Separator manufactured by Serono Diagnostics, Norwell, Mass. the DYNAL MPC-1 manufactured by DYNAL, Inc., Great Neck, N.Y. and the BioMag Separator, manufactured by Advanced Magnetics, Inc., Cambridge, Mass. A similar magnetic separator, manufactured by Ciba-Corning Medical Diagnostics, Wampole, Mass. is provided with rows of bar magnets arranged in parallel and located at the base of the separator. This device accommodates 60 test tubes, with the closed end of each tube fitting into a recess between two of the bar magnets.
The above-described magnetic separators have the disadvantage that the magnetic particles tend to form several layers on the inner surface of the sample container where they are entrapped along with impurities that are difficult to remove even with vigorous washing.
Colloidal magnetic materials are not readily separable from solution as such, even with powerful electro-magnets but, instead, require high gradient field separation techniques. See, R.R. Oder, IEEE Trans. Magnetics, 12:428-35 (1976); C. Owen and P. Liberti, Cell Separation: Methods and Selected Applications, Vol. 5, Pretlow and Pretlow eds., Academic Press, N.Y., (1986). The gradient fields normally used to filter such materials generate huge magnetic forces. Another useful technique for performing magnetic separations of colloidal magnetic particles from a test medium, by various manipulations of such particles, e.g., addition of agglomerating agents, is the subject of co-pending and commonly owned U.S. Pat. application Ser. No. 389,697, filed Aug. 4, 1989.
A commercially available high gradient magnetic separator is the MACS device made by Miltenyi Biotec GmbH, Gladbach, West Germany, which employs a column filled with a non-rigid steel wool matrix in cooperation with a permanent magnet. In operation, the enhanced magnetic field gradient produced in the vicinity of the steel wool matrix attracts and retains the magnetic particles while the non-magnetic test medium passes through and is removed from the column.
It has been found that the steel wool matrix of such prior art HGMS devices often gives rise to non-specific entrapment of biological entities other than the target substances which cannot be removed completely without extensive washing and resuspension of the particles bearing the target substance. Moreover, the size of the column in many of the prior art HGMS devices requires substantial quantities of experimental materials, which pose an impediment to their use in performing various useful laboratory-scale separations. In addition, the steel wool matrix may be harmful to certain sensitive cell types.
Although HGMS affords certain advantages in performing medical or biological analyses based on biospecific affinity reactions involving colloidal magnetic particles, the systems developed to date have not been entirely satisfactory for the above-mentioned reasons. Accordingly, it would be desirable to provide HGMS apparatus and methods which are of relatively simple construction and operation and yet maximize magnetic field gradients, and which reduce entrapment of non-target substances, eliminate loss of immobilized target substance due to shear forces or collisions with other biological entities, and employ standard microtiter plate wells, and the like, so as to be of practical utility in conducting various laboratory-scale separations, particularly in immunoassays and cell sorting.